The following discussion of the prior art is provided to place the invention in an appropriate technical context and enable the advantages of it to be more fully understood. It should be appreciated, however, that any discussion of the prior art throughout the specification should not be considered as an express or implied admission that such prior art is widely known or forms part of common general knowledge in the field.
The ability to determine DNA nucleotide sequences has become increasingly important in recent times. Previously, the two most commonly used methods for DNA sequencing are the enzymatic chain-termination method and the chemical cleavage technique, which both rely on gel electrophoresis to resolve, according to their size,
DNA fragments produced from a larger DNA segment. The electrophoresis step and the detection of the separated DNA-fragments are cumbersome procedures. However, whilst automated electrophoresis units are commercially available, electrophoresis is not well suited for large-scale genome projects or clinical sequencing where relatively cost-effective units with high throughput are needed. Thus, the need for non-electrophoretic methods for sequencing is significant.
Techniques enabling the rapid detection of a single DNA base change are also important tools for genetic analysis. A mini-sequencing protocol based on a solid phase principle was described previously, wherein the incorporation of a radio labelled nucleotide was measured and used for analysis of the three-allelic polymorphism of the human apolipoprotein E gene. However, radioactive methods are not well suited for routine clinical applications and hence the development of a simple non-radioactive method for rapid DNA sequence analysis has also been of interest.
Methods of sequencing based on the concept of detecting inorganic pyrophosphate (PPi) which is released during a polymerase reaction have been described previously (see International PCT Publication No.'s WO 93/23564 and WO 89/09283) and commonly referred to as pyrosequencing. As each nucleotide is added to a growing nucleic acid strand during a polymerase reaction, a pyrophosphate molecule is released. It has been found that pyrophosphate released under these conditions can be detected enzymically e.g. by the generation of light in the luciferase-luciferin reaction. Such methods enable a base to be identified in a target position and DNA to be sequenced simply and rapidly whilst avoiding the need for electrophoresis and the use of harmful radiolabels.
Early prior art methods for conducting pyrosequencing employed a 0.2 mL microcentrifuge tube (or similar) with reagents being added to the tube sequentially to detect the sequence of the DNA present in the tube. Whilst this method is relatively simple, the method suffers from the drawback that the read lengths are short, since the reaction is diluted with each addition of nucleotide reagent and/or reaction by-products are accumulated and the reaction conditions reach a point where the reaction no longer proceeds. For example, typically only about 80 bases can be sequenced reliably with this method.
Commercial equipment which utilise pyrosequencing have also been developed. These systems use flow cells to perform hybridisation of a target DNA/RNA molecule. To explain, single-stranded DNA is immobilised on a stationary bead which is positioned in the flow cell, typically by immobilising a double-stranded DNA and denaturing the complementary strand. Reagents, including a nucleotide (A, G, C, or T) are flowed past the bead and light is detected if a nucleotide is incorporated. The signal strength of the light is proportional to the number of nucleotides incorporated in a single reaction. Between exposing the bead to different nucleotides a wash step is also performed and the process is repeated to detect incorporation of the next nucleotide.
Other methods of sequencing by synthesis are also known, for example by using fluorescently-labelled nucleotides. In such a method DNA samples are first fragmented and the DNA double-helix is melted into single strands. The single DNA molecules are captured on a surface within a flow cell and serve as templates for the sequencing-by-synthesis process. Fluorescently-labelled nucleotides are added one at a time and incorporated into the growing complementary strand by a DNA polymerase enzyme. Unused nucleotides are washed away. Upon illumination with a laser, the incorporated nucleotides emit light that is detected. The fluorescent label is removed before the next nucleotide is added to continue the cycle. Tracking nucleotide incorporation determines the exact sequence of each individual DNA molecule.
Sequencing by ligation is also known. This DNA sequencing method uses the enzyme DNA ligase to identify the nucleotide present at a given position in a DNA sequence. The mismatch sensitivity of a DNA ligase enzyme is used to determine the underlying sequence of the target DNA molecule. See for example U.S. Pat. Nos. 5,750,341 and 4,883,750.
What is needed is apparatus for conducting assays and analyses, which can be used with a variety of chemistries and detection methods, and in particular for conducting assays that involve multiple reaction and washing steps such as used in sequencing nucleic acid. Further, what is needed is apparatus which can be used as a convenient replacement for assays which require a flow-through environment, or to replace fixed reaction vessel assays where, in case of nucleic acid sequencing, dilution effects limit the maximum sequencing read length.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the abovementioned prior art, or to provide a useful alternative.